Aloeferon isolation, manufacturing and its applications

ABSTRACT

The present invention is directed to a substantially pure therapeutically active aloe isolate having a molecular weight of about 70,000 and a dialysis method for making said aloe isolate.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 625,521 filed June28, 1984, now abandoned.

FIELD OF THE INVENTION

The present invention relates to a therapeutically active substanceisolated from aloe plants and methods for the preparation and usethereof.

BACKGROUND OF THE INVENTION

It has long been known that products of the aloe plant, particularly,aloe vera L. (aloe barbadensis Miller) are useful as medicaments in thetreatment and healing of a wide variety medical disorders. From ancienttimes, to the present, aloe gel has been reported to be useful in thetreatment of medical disorders in almost every field of medicine frompsychiatry to oncology, e.g., insomnia, insect bites and stings,infections, herpes, rheumatism, etc., and aloe is known to beparticularly effective in treating skin-related disorders.

Many attempts to identify and isolate therapeutically active componentsfrom aloe plants have been made in the prior art. For example, U.S. Pat.No. 3,103,466 is directed to medicaments useful for treating surfacewounds that include a polyuronide isolated from aloe gel which comprisesa polysaccharide having chemically bonded thereto one or more hexuronicacid radicals having a molecular weight from about 374,000 to 275,000 orthe non-toxic salts thereof.

U.S. Pat. No. 3,362,951 is directed to a therapeutically activepolysaccharide derived from the juice of aloe plants by mixing the juicewith a dilute aqueous solution of phosphomolybdic acid; separating theresulting precipitate from the aqueous solution; mixing a loweraliphatic polar solvent with the aqueous solution; separating theresulting blue-green precipitate therefrom and adding aqueoushypochlorous acid until the precipitate turns essentially white andrecovering the white precipitate which when washed with a water solublepolar solvent is a highly pure polysaccharide in the form of longpolymer chains (molecular weight about 420,000 to 520,000) comprised ofrepeating units containing substantially equal amounts of glucose andmannose residues, a small proportion of glucuronic acid residue andchemically bound calcium.

U.S. Pat. No. 4,225,486 is directed to a medicinally useful glycoproteinisolated from aloe plants and having a molecular weight of about 18,000,a protein to sugar ratio of 8 to 2 by weight and other specifiedproperties. The glycoprotein is prepared by precipitating a fractionfrom aloe juice with ammonium sulfate; dialyzing the precipitate toremove the ammonium sulfate; acidifying the dialyzed solution andcollecting the precipitate; dissolving the precipitate in buffer andseparating the protein product on a Sephadex G-200 column.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide an economicalone-step method for isolating from aloe plants a substantially puretherapeutically active polysaccharide linked with nitrogen, sulfur andphosphorous and having a molecular weight of about 70,000; that does notinvolve the use of organic solvents, heat, chromatography or ionexchange, absorption or elution methods and that eliminates harmfulcomponents, e.g., oxalic acid.

It is another object of the present invention to provide an aloe isolatewhich has useful thereapeutic properties including the accelerationgrowth, multiplication and healing of cells.

It is another object of the present invention to provide a polymerpolysaccharide having bactericidal properties that can be stored withoutthe aid of preservatives and stabilizers.

It is another object of this invention to provide substantially purealoe isolate that includes the primary biologically active ingredient ofaloe gel and that has value in the treatment of gastrointestinaldisorders, tumors, various skin ailments and as an antidote for certainpoisons.

It is yet another object of the present invention to provide asubstantially pure aloe isolate suitable for use as an additive tocosmetics, pharmaceuticals, soaps, detergents and the like to amelioratethe effects of cell damage.

It is also an object of this invention to provide an assay for thepotency and strength of aloe gels that conforms with the FDArequirements for use of said gels in food, drug and cosmetic products.

SUMMARY OF THE INVENTION

The present invention provides a non-destructive method for preparing analoe isolate comprising the steps of separating a gel from an aloe plantand dialyzing the separated gel against water until the non-dialyzablefraction has a molecular weight of about 70,000; and collecting thenon-dialyzable fraction which is the aloe isolate of this invention.This method is non-destructive because it does not employ conditions orchemicals, such as acids and organic solvents, which cause changes inthe molecular structure of the natural aloe isolate.

The aloe isolate of the present invention, which may be prepared by theforegoing method, comprises a polysaccharide linked with nitrogen,sulfur and phosphorous and having the following elemental analysis:

    ______________________________________                                        C     about       38.60%  S       "   1.04%                                   H     "           6.20%   N       "   0.13%                                   O     "           53.80%  P       "   0.13%.                                  ______________________________________                                    

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, is an infra-red spectrogram of the aloe isolate of the presentinvention;

FIG. 2, is a HPLC chromatogram of the aloe isolate of the presentinvention;

FIG. 3, is a NMR spectrogram of the aloe isolate of the presentinvention; and

FIG. 4, is a bar graph illustrating the effectiveness of the aloeisolate of the present invention in enhancing cell growth compared tocommercially available aloe vera gels.

DETAILED DESCRIPTION OF THE INVENTION

The aloe isolate of the present invention may be prepared from the wholealoe plant, or parts thereof, e.g. whole leaf or gel extracted frommucilagineous parenchyma (inner or central zone) by squeezing, pressing,filleting or other extraction methods suitable for removing ligninwalled cells, connective tissues, fibers, lignified sturdy veins, andclusters of calcium oxalate raphides sand and substances responsible forrapid discoloration and accelerated decomposition of the gel. Thepreferred aloe plants for purposes of this invention are aloe vera linne(aloe barbadensis Miller) Fam. Liliaceae or any of the 500 or so speciesof aloe containing the polysaccharide of this invention. The gel has afirm consistency only in the fresh aloe leaf. During processing andpurification of the aloe leaves the thin walled cells are disrupted andremoved and the gel contained within becomes watery and slightlycolloidal. Any contamination with yellow juice ingredients(anthraquinones) must be avoided because such compounds may produceallergic reactions and are considered contaminants.

The aloe isolate of this invention may be identified by its infra redspectra which is characterized by strong absorbance peaks at 3400 and1720 cm⁻¹ and medium absorbance peaks at 2900, 1550 and 1420 cm⁻¹ asshown in FIG. 1.

A preferred method for quantitative analysis of the aloe isolate of thisinvention is high pressure liquid chromatography (HPLC) employing a C₁₈column, an acetonitrile-water mobile phase and photometric detection at200 nm. This method produced the single peak chromatogram of FIG. 2 andthe concentration of aloe isolate in a sample can be calculated from thearea under the peak. This HPLC method may be effectively used fordetermining the potency and purity of aloe gel and aloe isolate productsin accordance with FDA requirements.

The aloe isolate of this invention has the NMR spectra shown in FIG. 3which may be characterized as follows:

    ______________________________________                                        Chemical Shift                                                                              Spectral Pattern                                                                            Chemical Group                                    ______________________________________                                        20       ppm      sharp single  methyl                                        70       ppm      broad multiple                                                                              sugar residue                                 174      ppm      broad multiple                                                                              acetate                                       ______________________________________                                    

Aloe Vera L. plants each having an average weight of about 5 Kg. wereprocessed in accordance with the invention to prepare the desired aloeisolate. Before the extraction of the gel was begun, latex (red juice)was drained off by cutting their leaves near the basal end and bleedingthem. The leaves were then cut parallel to their base and the jelly likelayer was removed by filleting. Plants of about 5 kg produced about 2 kgof gel and single average leaves of about 400 grams produced about 180grams of gel. The separated gel was blended in a high speed mixer andfiltered through gauze twice and then through coarse filter paper undervacuum. The resulting cloudy, milky filtrate was dialyzed againstdistilled water for about fifteen (15) hours in an ultrafiltrationsystem (Amicon DC 10) using hollow fiber columns (DIAFLO, HIX 50-8 andHIP 100-200) having a diameter of 2.3 cm and length of 20.3 cm at aninlet pressure of about 10 to 50 PSI and operating temperature of about20° to 60° C. This first dialysis procedure removed the aloe vera gel'scomponents of less than about 10,000 molecular weight. Thenon-dialyzable fraction I which included the gel components having amolecular weight greater than about 10,000 was then dialyzed a secondtime in a hollow fiber column against distilled water for about twentyfour (24) hours. This second dialysis removed components of less thanabout 30,000 molecular weight. The non-dialyzable fraction II resultingfrom the second dialysis was lyophilized and produced two bands whensubjected to gel electrophorisis using polyacrylamide gel, indicatingthe presence of two major components. The non-dialyzable fraction II wasthen dialyzed for a third time in a hollow fiber column againstdistilled water for about 24 hours thereby removing components with amolecular weight less than about 70,000 producing a substantially pure,non-dialyzable fraction III. The non-dialyzable fraction III was thenconcentrated and lyophilized producing 0.18 grams of the aloe isolate ofthis invention which produces a single band in gel electrophorsis usingpolyacrylamide gel. A freeze dried portion of the aloe isolate showed asymmetrical, geometric lattice-like drying pattern. A high pressureliquid chromatogram (FIG. 3) of the aloe isolate produced a single peakindicating that only a single compound was present. Quantitativeanalysis of the aloe isolate indicated that it was approximately 95% bywt. polysaccharide having the elemental analysis given above at page 4from which the following emperical formula was deduced (C₃₂₂, H₆₂₀, N,S₃, O₃₃₆ P)₇.

Samples of the aloe isolate were hydrolized in 6N hydrochloric acid at110° C. for 24 hours and the resulting hydrozylates were qualitativelydetermined by paper and gas chromatography, which indicated the presenceof mannose, glucose, arabinose, rhamnose and galactose. The aloe isolatealso tested positive for carbohydrates and negative for protein by theninhydrin test.

Comparison of Aloe Vera Gel and Aloe Isolate Cell Growth Ability

Fibroblast skin cells were obtained in a frozen ampule which was thawedin a waterbath at 37° C. with shaking. The ampule was then transferredinto a beaker containing 70% ethanol and opened under ascepticconditions and its content transferred to a culture flask. The resultantcell suspension was diluted with an appropriate volume of culture medium(Dulbecco's Modified Eagle) and incubated at 37° C. to allow the cellsto attach to and proliferate on the glass surface. Approximately 300,000cells were inoculated.

Stock solutions of the aloe vera gel and the aloe isolate were preparedand diluted with the medium to obtain the desired concentrations.Sterile techniques were used throughout the experimental study. Theculture medium was decanted from the culture bottles containing the skinfibroblast cells and the cells were washed once with a maintenancemedium. Previously prepared serial dilutions of each of the test agentswere dispensed into a sufficient number of bottles of cells to providetwo bottles of each dilution of the test agents for test periods of 24,48 and 72 hours. At the same time, sufficient bottles of cells wereinoculated with maintenance medium to provide two control bottles foreach experimental period.

At the end of 24, 48 and 72 hours, two bottles of each dilution and twocontrol bottles were trypsinized with 3.0 ml of 0.25% trypsin solutionto remove the cells from the surfaces of the bottles. After treatmentwith Trypsin for 15 minutes, the Trypsin solution containing the cellswas pipetted into 15 ml screw-cap tubes and centrifuged for 5 minutes.The liquid was decanted from the cells, and the cells were resuspendedin medium and again centrifuged for 5 minutes, 0.3 ml of 0.5% Trypanblue stain was then added to the suspension of cells in the maintenancemedium. After the cells were exposed to this stain for 3 minutes, theywere re-suspended by aspiration with a pipette. Aliquots were placed inthe counting chamber of Hemacytometer, and the cells were counted. Thenumber of cells in six aliquots from each bottle was counted and thetotal number of viable cells was determined. The non-viable cells werestained blue while the viable cells remained unstained and appearedcolorless. It can be seen from the data presented in FIG. 4 that thealoe isolate of this invention is far superior to aloe vera gel inenhancing cell growth.

Preliminary experiments were also conducted to determine the relativetoxicity of each of the test agents to the cells. Cells growing in glassprescription bottles were exposed to various dilutions of the test agentsolutions for time intervals of up to 72 hours to determine the highestconcentration of the test solutions that could be used without causingcomplete cell destruction. Preliminary qualitative observations revealedthat the 0.1% dilution of the gel was relatively toxic during early timeperiods and completely toxic at the end of 72 hours.

It has also been discovered that the aloe isolate of this invention andaloe vera gel itself act as antidotes to certain poisons, i.e., curareand atropine. Mice injected with about 10 to 25 mg per kg body weight ofthe aloe isolate of this invention showed better survival rate thancontrol mice when poisoned.

What is claimed is:
 1. A substantially pure pharmaceutically active aloe isolate comprising:a polysaccharide including nitrogen, sulfur and phosphorous and having a molecular weight of about 70,000; having an infra red spectra with principal peaks at about 3400, 2900, 1720, 1550 and 1420 cm⁻¹ ; and an NMR spectrum with absorbtion peaks at about 20 ppm (singlet) 70 ppm (broad multiplet) and 174 ppm (broad multiplet).
 2. The substantially pure pharmaceutically active aloe isolate according to claim 1, wherein the polysaccharide has the following elemental analysis C, 38.60%; H, 6.20%; O, 53.80%; S, 1.04%; N, 0.13% and P, 0.13%.
 3. The substantially pure pharmaceutically active aloe isolate according to claim 1 derived from an aloe plant selected from the group consisting of aloe vera linne and aloe barbadensis.
 4. A method for preparing a substantially pure aloe isolate, comprising the steps of:(A) separating aloe gel from an aloe plant; (B) dialyzing the gel against water; and (C) repeating step B to produce an aloe isolate comprising a polysaccharide including nitrogen, sulfur and phosphorous and having a molecular weight of about 70,000;having an infra red spectra with principal peaks at about 3400, 2900, 1720, 1550 and 1420 cm⁻¹ ; and an NMR specturm with absorbtion peaks at about 20 ppm (singlet), 70 ppm (broad multiplet) and 174 ppm (broad multiplet).
 5. The method according to claim 6 wherein the aloe plant selected from the group consisting of aloe vera linne and aloe barbadensis. 